Conversion of sugars to ionic liquids

ABSTRACT

Disclosed herein are methods for preparing sugar compositions. The methods include: i) forming a mixture including polysaccharide biomass and an ionic liquid solution, wherein the ionic liquid solution contains water and an ionic liquid, and the ionic liquid contains a) a cation and b) a sugar acid anion or a ketoacid anion; ii) maintaining the mixture under conditions sufficient to dissolve at least a portion of the polysaccharide present in the polysaccharide biomass; iii) adding at least one glycoside hydrolase to the mixture; and iv) maintaining the mixture containing the glycoside hydrolase under conditions sufficient to hydrolyze at least a portion of the dissolved polysaccharide, thereby forming the sugar compositions. The sugar compositions contain at least one monosaccharide or oligosaccharide. New sugar-based ionic liquids are also described.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application is a division of U.S. patent application Ser.No. 16/308,440, filed Dec. 7, 2018, which is a national stage entryunder 35 U.S.C. § 371 of International Pat. Appl. No. PCT/US2017/036438,filed Jun. 7, 2017, which claims priority to U.S. Provisional Pat. Appl.No. 62/346,955, filed on Jun. 7, 2016, which applications areincorporated herein by reference in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Contract No.DE-AC02-05CH11231 awarded by the United States Department of Energy. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

In recent years, tremendous efforts have been made to develop biofuelsmade from lignocellulosic biomass, which is derived from agriculturalwastes, forest residues, and dedicated energy crops. However, one of thegreatest limitations facing the economic viability of this technology isthe recalcitrant nature of the lignocellulosic biomass to enzymatichydrolysis into its component sugars. This resistance to breakdownnecessitates the use of pretreatment steps to enhance the accessibilityto and hydrolysis of the carbohydrate components present in thelignocellulosic biomass. Most pretreatment processes are thermo-chemicalprocesses that utilize combinations of high temperatures and pressures,or dilute acids or alkalis, to open up the structure of the biomass.Such processes necessitate the use of specialized equipment andhigh-energy inputs.

Ionic liquids (ILs) have come into prominence over recent years and havebeen used as innovative fluids for chemical processing. They are knownas environmentally friendly solvents primarily due to their lowvolatility and their potential recyclability. Recently, the use of ILsfor the pretreatment of biomass has been shown to be a promisingtechnology, allowing for the solubilization of crystalline cellulose andbiomass under relatively mild conditions. Reconstitution of the biomassfrom the IL results in an amorphous product that significantly increasesthe rate of enzymatic hydrolysis to its component soluble sugars. Forinstance, the IL 1-ethyl-3-methylimidazolium acetate [C₂Mim][OAc] hasbeen found to be effective at the dissolution of biomass and thesubsequent enhancement of enzymatic hydrolysis (also termedsaccharification).

The ionic liquid pretreatment process can generally be described as thedissolution of biomass into the ionic liquid at an elevated temperaturewith stirring, followed by the optional addition of a precipitant (or,alternatively, an anti-solvent) that precipitates the biomass fromsolution. This precipitant or anti-solvent is typically either water orethanol, or some other solvent with hydrogen bonding capacity. Once thebiomass has been precipitated, solid/liquid separation and downstreamenzymatic hydrolysis of the now amorphous biomass results inmonosaccharides suitable for fermentation. Typically, the ionic liquidpretreatment process employs atmospheric pressure and temperaturesranging from about 120° C. to 160° C. Recycling of ionic liquid can beachieved by distillation of the precipitating solvent.

Although pretreatment with ionic liquids has met with success, ionicliquids are expensive and the pretreatment process is both energy andtime intensive. Furthermore, ionic liquids can destabilize cellulasesused for hydrolysis and inhibit the growth of microorganisms used forsubsequent fermentation of the component soluble sugars. As such, whatis needed are methods for processing biomass in which pretreatment,hydrolysis, and fermentation steps are compatible with each other. Useof ionic liquids that are renewably sourced and non-toxic areparticularly desired. The present invention provides methods thatfulfill these and other needs.

BRIEF SUMMARY OF THE INVENTION

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, the claims andthe following embodiments:

-   -   1. A method for preparing a sugar composition, the method        comprising:    -   i) forming a mixture comprising polysaccharide biomass and an        ionic liquid solution, wherein    -   the ionic liquid solution comprises water and an ionic liquid,        and the ionic liquid comprises a) a cation and b) a sugar acid        anion or a ketoacid anion;    -   ii) maintaining the mixture under conditions sufficient to        dissolve at least a portion of the polysaccharide present in the        polysaccharide biomass;    -   iii) adding at least one glycoside hydrolase to the mixture; and    -   iv) maintaining the mixture containing the glycoside hydrolase        under conditions sufficient to hydrolyze at least a portion of        the dissolved polysaccharide, thereby forming the sugar        composition;

wherein the sugar composition comprises at least one monosaccharide oroligosaccharide.

2. The method of embodiment 1, wherein the sugar acid is selected fromthe group consisting of an aldaric acid, an aldonic acid, a uronic acid,and combinations thereof.

3. The method of embodiment 1 or embodiment 2, wherein the sugar acid isselected from the group consisting of mucic acid, saccharic acid,xylaric acid, arabinaric acid, and mannaric acid.

4. The method of embodiment 1, wherein the ketoacid is selected from thegroup consisting of α-ketoglutaric acid, pyruvic acid, and levulinicacid.

5. The method of embodiment 1, wherein the anion is selected from thegroup consisting of a mucic acid anion, a mucic acid dianion, anα-ketoglutaric acid anion, and an α-ketoglutaric acid dianion.

6. The method of any one embodiments 1-5, wherein the cation is selectedfrom choline, (C₁₋₁₈ alkyl)₃NH⁺, (C₁₋₆ alkyl)_(x)(C₆₋₁₈ alkyl)_(y)N⁺,(C₁₋₁₀ alkyl)_(z)imidazolium, (C₁₋₁₀ alkyl)_(z)pyrazolium, and mixturesthereof; wherein subscript x and subscript y are each 0, 1, 2, 3, or 4,and the sum of x and y is 4; and wherein each subscript z is 1, 2, or 3.

7 The method of embodiment 6, wherein the cation is choline.

8. The method of embodiment 6, wherein the cation is selected from1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium,1-ethyl-2,3-dimethylimidazolium, tris(2-hydroxyethyl)methylammonium,1-methylimidazolium, 1,2,4-trimethylpyrazolium, triethylammonium,tributylmethylammonium, hexadecyltrimethylammonium,myristyltrimethylammonium, tridodecylmethylammonium,trimethyloctadecylammonium, and choline.

9. The method of any one of embodiments 1-8, wherein the ionic liquidsolution comprises from about 25% (w/w) to about 95% (w/w) water.

10. The method of embodiment 1, wherein the ionic liquid solutioncomprises 50-90% (w/w) water; and further comprises 10-50% dicholinemucate (w/w) or 10-50% dicholine α-ketoglutarate (w/w).

11. The method of any one of embodiments 1-10, wherein the mixture instep i) comprises from about 5% (w/w) to about 30% (w/w) polysaccharidebiomass.

12. The method of any one of embodiments 1-11, wherein step ii)comprises maintaining the mixture of step i) at a temperature of atleast about 100° C. for at least about 30 minutes.

13. The method of any one of embodiments 1-12, wherein the pH of themixture in step i) is greater than 7.

14. The method of embodiment 13, further comprising:

ii-b) reducing the pH of the mixture containing the dissolvedpolysaccharide to less than 7 prior to adding the glycoside hydrolase instep iii).

15. The method of embodiment 14, wherein step ii-b) comprises adding anacid to the mixture resulting from step ii).

16. The method of embodiment 15, wherein the acid used in step ii-b) isthe same sugar acid or ketoacid used in step i).

17. The method of embodiment 15 or embodiment 16, wherein the molaramount of acid in step ii-b) is equal to the molar amount of the sugaracid or ketoacid in step i).

18. The method of any one of embodiments 15-17, wherein step ii-b)comprises adding an acid to the mixture resulting from step ii); whereinthe acid used in step ii-b) is the same sugar acid or ketoacid used instep i); and wherein the molar amount of acid in step ii-b) is equal tothe molar amount of the sugar acid or ketoacid in step i).

19. The method of embodiment 1, wherein the polysaccharide biomasscomprises cellulose, hemicellulose, lignocellulose, or mixtures thereof.

20. The method of embodiment 19, wherein the polysaccharide biomasscomprises lignocellulose.

21. The method of any one of embodiments 1-20, wherein thepolysaccharide biomass is derived from corn stover, corn fiber,hardwood, softwood, cereal straw, switchgrass, Miscanthus, rice hulls,municipal solid waste (MSW), industrial organic waste, office paper, ormixtures thereof.

22. The method of any one of embodiments 1-21, wherein the glycosidehydrolase is a cellulase.

23. The method of any one of the preceding embodiments 1-22, wherein theglycoside hydrolase is selected from the group consisting of anendoglucanase, an exoglucanase, a β-glucosidase, a xylanase, andmixtures thereof.

24. An ionic liquid comprising at least one sugar acid anion and atleast one cation, wherein the anion is selected from the groupconsisting of a sugar acid anion and a ketoacid ion.

25. The ionic liquid of embodiment 24, wherein the sugar acid isselected from the group consisting of an aldaric acid, an aldonic acid,a uronic acid, or a combination thereof.

26. The ionic liquid of embodiment 24 or embodiment 25, wherein thesugar acid is selected from the group consisting of mucic acid,saccharic acid, xylaric acid, arabinaric acid, and mannaric acid.

27. The ionic liquid of claim 24, wherein the ketoacid is selected fromthe group consisting of α-ketoglutaric acid, pyruvic acid, and levulinicacid.

28. The ionic liquid of any one of embodiments 24-27, wherein the anionis selected from the group consisting of a mucic acid anion, a mucicacid anion, an α-ketoglutaric acid anion, and an α-ketoglutaric aciddianion.

29. The ionic liquid of any one of embodiment 24-28, wherein the cationis selected from choline, (C₁₋₁₈ alkyl)₃NH⁺, (C₁₋₆ alkyl)_(x)(C₆₋₁₈alkyl)_(y)N⁺, (C₁₋₁₀ alkyl)_(z)imidazolium, (C₁₋₁₀ alkyl)_(z)pyrazolium,and mixtures thereof; wherein subscript x and subscript y are each 0, 1,2, 3, or 4, and the sum of x and y is 4; and wherein each subscript z is1, 2, or 3.

30. The ionic liquid of embodiment 29, wherein the cation is choline.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of sugar yield resulting from one-potpre-treatment and saccharification of switch grass using an aldaricacid-based choline ionic liquid, [Ch]₂[Mu], and using [Ch]₂[glutamate].

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a composition comprising an ionicliquid (IL) derived from a sugar, such as an aldaric acid converted fromsugar. In some embodiments, the composition comprises a mixture of ILsderived from a sugar or sugars. In some embodiments, the sugar isderived from pretreatment and/or saccharification of biomass.

Sugars derived from lignocellulose can be converted into ILs byderivitization into aldaric acids. A variety of sugar-derived anions andcations can be used to form ILs that can be used for efficient biomasspretreatment.

The present invention provides for a method to convert a monomeric sugarinto ionic liquid anions and/or cations. In some embodiments the methodcomprises converting a mixture of monomeric sugars into ionic liquidanions and/or cations. The monomeric sugars can be derived fromlignocellulosic biomass hydrolysates rich in glucose and xylose or fromany other sugar present in biomass, such as galactose, arabinose,mannose, or the like, or a mixture thereof. In some embodiments, themonomeric sugar is a C₆ sugar or a C₅ sugar, or a mixture thereof. Insome embodiments, the monomeric sugar is a glucose, xylose, galactose,arabinose, mannose, or the like, or a mixture thereof.

To generate anions, a sugar can be chemically or enzymatically convertedto its uronic, aldonic, or aldaric acid form. These anions can be pairedwith other non-sugar derived IL cations, such as choline,1-ethyl-3-methylimidazolium, or the like, or with sugar cationsgenerated by forming positively charged salts, using processes such asamination. It is demonstrated that the aldaric acid of galactose, mucicacid, can form an IL that is effective at pretreating lignocellulosicbiomass when paired with a choline cation. A 36 wt. % choline:mucic acidIL generated from a mixture of choline hydroxide and mucic acid titratedto a pH of 13 is used to pretreat switchgrass at 140° C. for lhr, thenthe pH was adjusted to pH 5 and the IL concentration to 20%. This slurryis then enzymatically hydrolyzed with a commercial cellulase cocktail at10 mg enzyme/g glucan and liberated 76% glucose and 49% xylose.

The process can be used in lignocellulosic biorefineries.Lignocellulosic biomass is pretreated and hydrolyzed, producing amixture of sugars primarily consisting of glucose and xylose. A portionof the hydrolysate can be removed and used to convert the glucose,xylose, or both into their respective sugar acids (e.g., aldaric acidsand the like). A number of known processes can be used for convertingthe sugars to sugar acids as described, for example, in U.S. Pat. Appl.Pub Nos. US 2015/0065749, US 2012/0045804, and US 2008/0187984, as wellas U.S. Pat. Nos. 7,982,031 and 6,518,419, which are incorporated hereinby reference. These anions can then be paired with an IL cation, such ascholine at high pH (≥10) in water and subsequently used to pretreatadditional lignocellulosic biomass, or be sold for other purposes.Sugar-derived cations could also be generated as well and used to formILs to be used for pretreatment or applications.

I. Definitions

As used herein, the term “sugar composition” refers to a mixturecontaining one or more monosaccharides, oligosaccharides, orcombinations thereof. Sugar compositions prepared according to themethods of the invention are also referred to as “hydrolysates” in thepresent application.

As used herein, the term “monosaccharide” refers to a sugar having afive-membered carbon backbone (i.e., a pentose) or a six-membered carbonbackbone (i.e., a hexose). Examples of monosaccharides include, but arenot limited to, glucose, ribose, fucose, xylose, arabinose, galactose,mannose, glucuronic acid, and iduronic acid. Monosaccharides alsoinclude pentoses and hexoses substituted with hydroxy groups, oxogroups, amino groups, acetylamino groups, and other functional groups.

As used herein, the term “oligosaccharide” refers to a compoundcontaining at least two sugars covalently linked together.Oligosaccharides include disaccharides, trisaccharides,tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides,octasaccharides, and the like. Covalent linkages for linking sugarsgenerally consist of glycosidic linkages (i.e., C—O—C bonds) formed fromthe hydroxyl groups of adjacent sugars. Linkages can occur between the1-carbon (the anomeric carbon) and the 4-carbon of adjacent sugars(i.e., a 1-4 linkage), the 1-carbon (the anomeric carbon) and the3-carbon of adjacent sugars (i.e., a 1-3 linkage), the 1-carbon (theanomeric carbon) and the 6-carbon of adjacent sugars (i.e., a 1-6linkage), or the 1-carbon (the anomeric carbon) and the 2-carbon ofadjacent sugars (i.e., a 1-2 linkage). Other linkages can be present inthe oligosaccharide, depending on the particular sugar subunits present.Those of skill in the art will appreciate that a sugar can be linkedwithin an oligosaccharide such that the glycosidic bond at the anomericcarbon is in the α- or β-configuration.

As used herein, the term “polysaccharide” generally refers to a compoundcontaining 10 or more sugars linked together as described foroligosaccharides.

As used herein, the term “biomass” and “polysaccharide biomass” are usedinterchangeably to refer to plant-based material that includes aplurality of components such as lignin, cellulose, and hemicellulose.Sources of biomass includes trees, shrubs, grasses, wheat, wheat straw,sugar cane bagasse, corn, corn husks, corn kernel including fiber fromkernels, products and by-products from milling of grains such as corn,rice, wheat, and barley, as well as municipal solid waste, waste paper,and yard waste. Biomass sources can also include herbaceous material,agricultural residues, forestry residues, and paper mill residues.Additional examples include branches, bushes, canes, corn and cornhusks, energy crops, forests, fruits, flowers, grains, grasses,herbaceous crops, leaves, bark, needles, logs, roots, saplings, shortrotation woody crops, shrubs, switchgrasses, trees, vegetables, fruitpeels, vines, sugar beet pulp, wheat midlings, oat hulls, hard and softwoods, organic waste materials generated from agricultural processesincluding farming and forestry activities, or mixtures thereof.

As used herein, the term “lignocellulosic biomass” refers to naturaland/or synthetic materials containing lignin, cellulose, and/orhemicellulose. Generally, these materials also contain (but need notcontain) xylan, protein, and/or other carbohydrates, such as starch.

As used herein, the term “cellulose” refers to refers to a homopolymerof β(1→4) linked D-glucose units that form a linear chain. Cellulose cancontain several hundred to several thousand or more glucose units,making cellulose a polysaccharide.

As used herein, the term “hemicellulose” refers to a heteropolymercontaining different saccharide units, including but not limited to,xylose, mannose, galactose, rhamnose and arabinose. Hemicellulose formsa branched polymer with several hundred to several thousand sugar units.Hemicellulose can include both pentose and hexose sugars.

As use herein, the term “lignin” refers to a phenylpropane polymer ofmonolignol monomers (p-coumaryl alcohol, coniferyl alcohol, and sinapylalcohol) found as an integral part of the secondary cell walls of plantsand certain types of algae.

As used herein, the term “ionic liquid” refers to an organic salt thatis a liquid at room temperature rather than a solid or crystallinesubstance. Ionic liquids typically exhibit a number of advantageousproperties, including low volatility, thermal stability, and the abilityto dissolve a wide range of solutes under mild conditions.

As used herein, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. Alkyl can include any number of carbons, such as C₁₋₂, C₁₋₃,C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄,C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. For example, C₁₋₆ alkyl includes, butis not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can alsorefer to alkyl groups having up to 20 carbons atoms, such as, but notlimited to heptyl, octyl, nonyl, decyl, etc. An “alkane” refers to theparent compound of the alkyl radicals described herein.

As used herein, the term “alkenyl” refers to a straight chain orbranched hydrocarbon having at least 2 carbon atoms and at least onedouble bond. Alkenyl can include any number of carbons, such as C₂,C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆,C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆. Alkenyl groups can have any suitablenumber of double bonds, including, but not limited to, 1, 2, 3, 4, 5 ormore. Examples of alkenyl groups include, but are not limited to, vinyl(ethenyl), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl,1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl,1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. An“alkene” refers to the parent compound of the alkenyl radicals describedherein.

As used herein, the term “sugar acid” refers to a monosaccharide asdescribed herein having at least one two carboxy moiety (i.e., at leastone —COOH group). As used herein, the term “sugar acid anion” refers toa sugar acid wherein at least one of the carboxy moieties isdeprotonated (i.e., present as a —COO anion). Sugar acid anions aregenerally bound to cations in an ionic liquid via electrostaticinteraction.

As used herein, the term “ketoacid” refers to a an alkane or alkenehaving at least one carboxy moiety and least one oxo moiety (i.e., atleast one —COOH group and at least one C═O group). As used herein, theterm “ketoacid anion” refers to a ketoacid wherein at least one of thecarboxy moieties is deprotonated (i.e., present as a —COO anion).Ketoacid anions are generally bound to cations in an ionic liquid viaelectrostatic interaction.

As used herein, the term “cation” refers to a positively chargedmolecule that pairs with an anion in an ionic liquid via electrostaticinteraction. Examples of cations suitable for inclusion in ionic liquidsinclude, but are not limited to, ammonium, imidazolium, pyridinium,sulfonium, and phosphonium cations.

As used herein, the term “molar ratio” refers to the number of moles ofone species in a mixture relative to the number of moles of a secondspecies in the mixture. As a non-limiting example, an ionic liquidhaving an anion:cation ratio of 1:2 has at least two moles of the cationfor every mole of the anion. For ionic liquids where the molar ratio ofthe anion to the cation is at least 1:2, the molar ratio can be, e.g.,1:2.1, 1:2.5, or 1:3, or 1:4.

As used herein, the term “choline” refers to the2-hydroxy-N,N,N-trimethylethan-amonium cation and salts thereof (e.g.,2-hydroxy-N,N,N-trimethylethanamonium hydroxide). The term “dicholinemucate,” also referred to as [Ch]₂[Mu], refers to an ionic liquid havingmucic acid dianions and two choline cations for each of the mucic aciddianions. As used herein, the term “dicholine α-ketoglutarate,” alsoreferred to as [Ch]₂[α-Kg], refers to an ionic liquid havingα-ketoglutaratic acid dianions and two choline cations for each of theα-ketoglutaratic acid dianions.

As used herein, the term “pH” refers to refers to a measurement of theconcentration of hydrogen ions in a mixture such as an aqueous solution.pH is expressed as the decimal logarithm (i.e., log₁₀) of the reciprocalof the hydrogen ion concentration in the mixture. The pH of a mixturecan be determined using a number of known techniques. One of skill inthe art will know how to adjust the pH of a mixture by adding acidsand/or bases to the mixture.

As used herein, the term “acid” refers to a substance that is capable ofdonating a proton (i.e., a hydrogen cation) to form a conjugate base ofthe acid. Examples of acids include, but are not limited to,hydrochloric acid, sulfuric acid, acetic acid, and formic acid.

As used herein, the term “base” refers to a substance that is capable ofaccepting a proton (i.e., a hydrogen cation) to form a conjugate acid ofthe base. Examples of bases include, but are not limited to, sodiumhydroxide, potassium hydroxide, sodium bicarbonate, and potassiumcarbonate.

As used herein, the terms “dissolve” and “dissolution” refer to thesolvation of a solute with a solvent to form a solution. Moreparticularly, dissolution refers to the partial or completesolubilization of biomass in an ionic liquid or an ionic liquidsolution. In the methods of the invention, dissolution oflignocellulosic biomass can include partial or complete disruption ofintra- and intermolecular hydrogen bonds present in cellulose polymerchains, partial or complete disruption of interactions between celluloseand hemicellulose, and partial or complete solubilization of lignin.

The terms “hydrolyze,” “hydrolysis,” and “saccharification,” when usedherein with respect to polysaccharide chemistry, refer to the cleavageof one or more glycosidic bonds in an oligosaccharide or apolysaccharide by water. The hydrolysis is typically catalyzed by anenzyme such as a glycoside hydrolase. Hydrolysis can also be promoted byaddition of a catalyst such as an acid or base.

As used herein, the term “glycoside hydrolase” refers to an enzyme thatcatalyzes the cleavage of the glycosidic linkage in oligosaccharides orpolysaccharides by water to release smaller sugars.

The terms “about” and “around,” as used herein to modify a numericalvalue, indicate a close range surrounding that explicit value. If “X”were the value, “about X” or “around X” would indicate a value from 0.9Xto 1.1X. “About X” thus includes, for example, a value from 0.95X to1.05X, or from 0.98X to 1.02X, or from 0.99X to 1.01X. Any reference to“about X” or “around X” specifically indicates at least the values X,0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X,1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.07X, 1.08X, 1.09X, and 1.10X.Accordingly, “about X” and “around X” are intended to teach and providewritten description support for a claim limitation of, e.g., “0.98X.”

II. Sugar-Based Ionic Liquids for Biomass Pretreatment andSaccharification

In one aspect, the present invention provides a method for preparing asugar composition. In typical embodiments, a method of the invention,includes:

-   -   i) forming a mixture including polysaccharide biomass and an        ionic liquid solution, wherein the ionic liquid solution        contains water and an ionic liquid, and the ionic liquid        contains a) a cation and b) a sugar acid anion or a ketoacid        anion;    -   ii) maintaining the mixture under conditions sufficient to        dissolve at least a portion of the polysaccharide present in the        polysaccharide biomass;    -   iii) adding at least one glycoside hydrolase to the mixture; and    -   iv) maintaining the mixture containing the glycoside hydrolase        under conditions sufficient to hydrolyze at least a portion of        the dissolved polysaccharide, thereby forming the sugar        composition;        wherein the sugar composition contains at least one        monosaccharide or oligosaccharide.

Polysaccharide Biomass

The methods of the invention are used for the production of sugarcompositions (containing monosaccharides, oligosaccharides, and/orpolysaccharides) as chemical or fermentation feedstocks from biomassmaterials. The feedstocks, in turn, can be used for the production ofethanol, plastics, or other products or intermediates. Biomass caninclude, but is not limited to, wood resources, municipal solid waste,wastepaper, and crop residues (see, for example, Wiselogel et al., 1995,in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-118,Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25:695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork). Other examples of biomass include, without limitation, crops suchas starch crops (e.g., corn, wheat, or barley), sugar crops (e.g.,sugarcane, energy cane or sugar beet), forage crops (e.g., grasses,alfalfa, or clover), and oilseed crops (e.g., soybean, sunflower, orsafflower); wood products such as trees, shrubs, and wood residues(e.g., sawdust, bark or the like from forest clearings and mills); wasteproducts such as municipal solid waste (MSW; e.g., paper, food and yardwastes, or wood), process waste, and paper sludge; and aquatic plantssuch as algae, water weeds, water hyacinths, or reeds and rushes. Otherexamples of biomass include sorghum, rice hulls, rice straw, wheatstraw, and other straws.

Accordingly, some embodiments of the invention provide a method forpreparing a sugar composition as described above, wherein thepolysaccharide biomass comprises cellulose, hemicellulose,lignocellulose, or mixtures thereof. In some embodiments, thepolysaccharide biomass comprises lignocellulose.

Biomass materials typically contain a mixture of polysaccharide species.In many instances, the predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant ishemi-cellulose, and the third is pectin. The secondary plant cell wall,produced after the cell has stopped growing, also containspolysaccharides and is strengthened through polymeric lignin covalentlycross-linked to hemicellulose.

Cellulose is a homopolymer of anhydrocellobiose and thus a linearβ-(1-4)-D-glucan, while hemicelluloses include a variety of sugarsubunits, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which helps stabilize the cell wall matrix.

In addition to the polysaccharides described above, polysaccharidebiomass typically contains lignin. Lignin is a phenylpropane polymer ofmonolignol monomers. It is generally found as an integral part of thesecondary cell walls of plants and certain types of algae. There arethree monolignol monomers, methoxylated to various degrees: p-coumarylalcohol, coniferyl alcohol, and sinapyl alcohol. These lignols areincorporated into lignin in the form of the phenylpropanoidsp-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), respectively.Gymnosperms have a lignin that consists almost entirely of G with smallquantities of H. That of dicotyledonous angiosperms is more often thannot a mixture of G and S (with very little H), and monocotyledonouslignin is a mixture of all three. Many grasses have mostly G, while somepalms have mainly S. All lignins contain small amounts of incomplete ormodified monolignols, and other monomers are prominent in non-woodyplants. Unlike cellulose and hemicellulose, lignin cannot bedepolymerized by hydrolysis. Cleavage of the principal bonds in thelignin polymer generally proceeds through oxidation.

In some embodiments, the polysaccharide biomass is derived from cornstover, corn fiber, hard wood, softwood, cereal straw, switchgrass,Miscanthus, rice hulls, municipal solid waste (MSW), industrial organicwaste, office paper, or mixtures thereof.

Ionic Liquids

A number of ionic liquids can be used in the methods of the invention.In general, the ionic liquid is suitable for pretreatment of the biomassand compatible with glycoside hydrolases used for saccharification ofcellulose and other polysaccharides. The ionic liquids contain a sugaracid ion paired with cations via electrostatic interactions. In certainembodiments, the ionic liquid used for biomass pretreatment contains onesugar acid acid di-anion paired with two cations.

Any suitable sugar acid can be used in the methods of the invention. Thesugar acids can be derived from lignocellulosic biomass hydrolysatesrich in glucose and xylose or from any other sugar present in biomass,such as galactose, arabinose, mannose, or the like, or a mixturethereof. In some embodiments, the sugar acid is a C₆ sugar acid or a C₅sugar acid, or a mixture thereof.

In some embodiments, the sugar acid is selected from the groupconsisting of an aldaric acid, an aldonic acid, a uronic acid, andcombinations thereof. In some embodiments, the sugar acid is selectedfrom the group consisting of mucic acid [i.e.,(2S,3R,4S,5R)-2,3,4,5-tetrahydroxyhexanedioic acid; meso-galactaricacid]; saccharic acid [i.e.,(2S,3S,4S,5R)-2,3,4,5-tetrahydroxyhexanedioic acid; D-glucaric acid];D-xylaric acid [i.e., (2R,4S)-2,3,4-trihydroxypentanedioic acid];D-arabinaric acid [i.e., (2S,4S)-2,3,4-trihydroxypentanedioic acid], andmannaric acid [i.e., (2S,3S,4S,5S)-2,3,4,5-tetrahydroxyhexanedioicacid].

Any ketoacid can be used in the methods of the invention. Like sugaracids, ketoacids can be derived from lignocellulosic biomass. In someembodiments, the ketoacid is a C₆ ketoacid or a C₅ ketoacid, or amixture thereof. Examples of ketoacids for use in the ionic liquids andmethods of the invention include, but are not limited to, α-ketoglutaricacid, pyruvic acid, acetoacetic acid, levulinic acid, and the like.

In some embodiments, the anion in the ionic liquid is selected from thegroup consisting of a mucic acid anion, a mucic acid dianion, anα-ketoglutaric acid anion, and an α-ketoglutaric acid dianion.

The ionic liquids used in the methods of the invention can contain anysuitable cation. Suitable cations include, but are not limited to,ammonium cations and imidazolium cations. Examples of ammonium cationsinclude, but are not limited to, 2-hydroxyethyl-trimethylammonium,benzyldimethyltetradecylammonium, benzyltrimethylammonium,butyltrimethylammonium, choline, diethylmethyl(2-methoxyethyl)ammonium,ethyldimethylpropylammonium, methyltrioctadecylammonium,methyltrioctylammonium, tetrabutylammonium, tetradodecylammonium,tetraethyl ammonium, tetraheptylammonium, tetrahexadecylammonium,tetrahexylammonium, tetrakis(decyl)ammonium, tetramethylammonium,tetraoctylammonium, tributylmethylammonium, triethylmethylammonium, andtris(2-hydroxyethyl)methylammonium.

The imidazolium cations can be, but are not limited to,1-alkyl-3-alkylimidazolium cations, wherein an “alkyl” is an alkyl groupcomprising from 1 to 10 carbon atoms. In some embodiments, the “alkyl”is a methyl group, ethyl group or butyl group. Examples of imidazoliumcations include: 1-(2-hydroxyethyl)-3-methylimidazolium;1-(3-cyanopropyl)-3-methylimidazolium; 1,2,3-trimethylimidazolium;1,2-dimethyl-3-propylimidazolium; 1,3-bis(cyanomethyl)imidazolium;1,3-diethoxyimidazolium; 1,3-dihydroxy-2-methylimidazolium;1,3-dihydroxyimidazolium; 1,3-dimethoxy-2-methylimidazolium;1,3-dimethoxyimidazolium; 1,3-dimethylimidazolium;1-allyl-3-methylimidazolium; 1-benzyl-3-methylimidazolium;1-butyl-2,3-dimethylimidazolium; 1-butyl-3-methylimidazolium (BMIM);1-decyl-3-methylimidazolium; 1-dodecyl-3-methylimidazolium;1-ethyl-2,3-dimethylimidazolium (EDIM); 1-ethyl-3-methylimidazolium(EMIM); 1-hexyl-3-methylimidazolium; 1-methyl-3-octylimidazolium;1-methyl-3-propylimidazolium; 1-methylimidazolium (MIM); and4-(3-butyl-1-imidazolio)-1-butanesulfonate.

Other cations can be used in the ionic liquids of the present invention,including, but not limited to: pyridinium cations (e.g.,N-ethylpyridinium, N-butylpyridinium, and the like); sulfonium cations(e.g., trimethylsulfonium, triethylsulfonium, tributylsulfonium,diethylmethylsulfonium, dimethylpropylsulfonium, dimethylhexylsulfonium,and the like); and phosphonium cations (e.g., tetramethylphosphonium,tetraethylphosphonium, tetrapropylphosphonium, tetrabutylphosphonium,tetraoctylphosphonium, tetraphenylphosphonium,trimethylethylphosphonium, triethylmethylphosphonium,hexyltrimethylphosphonium, trimethyloctylphosphonium, and the like).

In some embodiments, the cation is selected from choline, (C₁₋₁₈alkyl)₃NH⁺, (C₁₋₆ alkyl)_(x)(C₆₋₁₈ alkyl)_(y)N⁺, (C₁₋₁₀alkyl)_(z)imidazolium, (C₁₋₁₀ alkyl)_(z)pyrazolium, and mixturesthereof; wherein subscript x and subscript y are each 0, 1, 2, 3, or 4,and the sum of x and y is 4; and wherein each subscript z is 1, 2, or 3.

The cation be, for example, (C₁₋₁₆ alkyl)₃NH⁺, (C₁₋₁₂ alkyl)₃NH⁺, (C₁₋₁₀alkyl)₃NH⁺, (C₁₋₈ alkyl)₃NH⁺, (C₁₋₆ alkyl)₃NH⁺, or (C₁₂₋₁₈ alkyl)₃NH⁺,(C₁₆₋₁₈ alkyl)₃NH⁺. The cation be (C₁₋₃ alkyl)_(x)(C₆₋₁₂ alkyl)_(y)N⁺ or(C₁₋₂ alkyl)_(x)(C₆₋₈ alkyl)_(y)N⁺, wherein x and subscript y are each0, 1, 2, 3, or 4, and the sum of x and y is 4. The cation can be (C₁₋₈alkyl),_(z) imidazolium, (C₁₋₆ alkyl)_(z)imidazolium, (C₁₋₈alkyl)_(z)pyrazolium, or (C₁₋₆ alkyl)_(z)pyrazolium, wherein eachsubscript z is 1, 2, or 3.

In some embodiments, the cation is selected from1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium,1-ethyl-2,3-dimethylimidazolium, tris(2-hydroxyethyl) methylammonium,1-methylimidazolium, 1,2,4-trimethylpyrazolium, triethylammonium,tributylmethylammonium, hexadecyltrimethylammonium,myristyltrimethylammonium, tridodecylmethylammonium,trimethyloctadecylammonium, and choline. In some embodiments, the cationis choline.

The ionic liquids used in the methods of the invention can be preparedby combining a sugar acid, or a salt thereof, with a salt containing thecation to be incorporated into the ionic liquid. The sugar acid and thecation can be combined as solutions in water or in a suitable organicsolvent. As a non-limiting example, one equivalent of mucic acid inaqueous solution can be combined with two equivalents of cholinehydroxide in aqueous solution. Water can be removed at elevatedtemperature and/or under reduced pressure. Water-miscible co-solvents,including but not limited to methanol, acetonitrile, acetone, and thelike, can be used to precipitate excess anions or cations for removal bycentrifugation or filtration. Impurities can be removed by passing theionic liquid through activated charcoal, polymeric ion-exchange resins,or other decolorizing agents.

In general, the molar ratio of the sugar acid anions in the ionic liquidsolution to the cations in the ionic liquid solution will be sufficientto provide a solution pH of at least about 7. In certain embodiments,the molar ratio of the sugar acid anion to the cation is at least about1:2. The molar ratio of the sugar acid anion to the cation can be, forexample, at least 1:1.8, or at least 1:1.9; or at least 1:2, or at least1:2.1 or at least 1:2.2. When the mixture of the sugar acid and the saltis made in aqueous solution, the pH of the resulting ionic liquidsolution will be basic. In general, the pH of the ionic liquid solutionis above 7. The pH of the ionic liquid solution can be, for example, atleast 7, at least 7.5, at least 8, at least 8.5, at least 9, at least9.5, at least 10, at least 10.5, at least 11, at least 11.5, at least12, at least 12.5, at least 13, or at least 13.5. In certainembodiments, one equivalent of a sugar acid or ketoacid is combined withtwo equivalents of a salt containing a cation and a basic anion. As anon-limiting example, combination of one equivalent of mucic acid withtwo equivalents of choline hydroxide in aqueous solution will result inan ionic liquid solution having a pH of about 13.5. One of skill in theart will appreciate that the pH of the ionic liquid solution will varydepending on the particular sugar acid and cation used, the ratio of thesugar acid and the cation, and their absolute concentrations.

As described in more detail below, the pH of mixture containing theionic liquid solution can be reduced after the pretreatment step so thatmixture is compatible with enzymes, such as cellulases, used to breakdown the pretreated biomass. In certain embodiments, the pH is reducedby adding the same sugar acid that is present in the ionic liquid.Accordingly, in some embodiments an ionic liquid solution having a pH ofat least about 10 (e.g., from about 10.5 to about 13.5) is obtained bycombining one equivalent of a sugar acid or ketoacid with twoequivalents of a salt containing a cation; the pH of the ionic liquidsolution is then reduced to below about 7 via addition of a secondequivalent of the acid prior to the introduction of enzymes such asglycoside hydrolases.

In some embodiments an ionic liquid solution having a pH greater than 7(e.g., at least 10) is obtained by combining one equivalent of mucicacid with two equivalents of choline hydroxide. After pretreatment ofthe polysaccharide biomass, the pH of the ionic liquid solution is thenreduced to below about 7 (e.g., around 5) via addition of a secondequivalent of mucic acid prior to the introduction of enzymes such asglycoside hydrolases.

In some embodiments an ionic liquid solution having a pH greater than 7(e.g., a pH of at least about 10) is obtained by combining oneequivalent of α-ketoglutaric acid with two equivalents of cholinehydroxide. After pretreatment of the polysaccharide biomass, the pH ofthe ionic liquid solution is then reduced to below about 7 (e.g., around5) via addition of a second equivalent of α-ketoglutaric acid prior tothe introduction of enzymes such as glycoside hydrolases.

The pH of an ionic liquid solution can be raised or lowered as necessaryby adding bases, such as sodium hydroxide or potassium hydroxide, andacids, such as hydrochloric acid or sulfuric acid, to the ionic liquidsolution. As a non-limiting example, combination of one equivalent ofmucic acid with one equivalent of choline hydroxide in aqueous solutionfollowed by the addition of potassium hydroxide can provide an ionicliquid having a pH of about 11.

The ionic liquid solution can contain any suitable amount of water. Ingeneral, the ionic liquid solutions used in the methods of the inventioncontain from about 0.1% water to about 95% water by weight of the ionicliquid solution. An ionic liquid solution can contain, for example, fromabout 5% to about 90% water, or from about 10% to about 80% water, orfrom about 20% to about 60% water, or from about 30% to about 50% water,or from about 0.1 to about 50% water, or from about 5% to about 45%water, or from about 10% to about 40% water, or from about 15% to about35% water, or from about 20% to about 30% water by weight of the ionicliquid solution. The ionic liquid solution can contain about 0.1, 1, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, or about 95% water by weight of the ionic liquid solution.

In certain embodiments, the ionic liquid solution contains from about50% (w/w) to about 95% (w/w) water. The ionic liquid solution cancontain from about 50% to about 55% water, or from about 55% to about60% water, or from about 60% to about 65% water, or from about 65% toabout 70% water, from about 70% to about 75% water, or from about 75% toabout 80% water, or from about 80% to about 85% water, or from about 85%to about 90% water, or from about 90% to about 95% water by weight ofthe ionic liquid solution. The ionic liquid solution can contain fromabout 70% to about 90% water, or from about 72% to about 85% water, orfrom about 73% to about 80% water by weight of the ionic liquidsolution. The ionic liquid solution can contain from about 70% to about95% water, or from about 80% to about 93% water, or from about 85% toabout 92% water by weight of the ionic liquid solution. In suchembodiments, the ionic liquid solution will contain from about 30% (w/w)to about 5% (w/w) ionic liquid. The ionic liquid solution can containfrom about 25% to about 30% ionic liquid, or from about 20% to about 25%ionic liquid, or from about 15% to about 20% ionic liquid, or from about10% to about 15% ionic liquid, or from about 5% to about 10% ionicliquid by weight of the ionic liquid solution. The ionic liquid solutioncan contain from about 10% to about 30% ionic liquid, or from about 15%to about 28% ionic liquid, or from about 20% to about 27% ionic liquidby weight of the ionic liquid solution. The ionic liquid solution cancontain from about 5% to about 30% ionic liquid, or from about 7% toabout 20% ionic liquid, or from about 8% to about 15% ionic liquid byweight of the ionic liquid solution.

Other amounts of water and ionic liquid can be used in the methods ofthe invention, depending in part on factors such as the type of biomassmaterial to be treated and the particular cations and anions to beincluded in the ionic liquid.

In some embodiments, the ionic liquid solution contains:

-   -   about 50-90% (w/w) water; and    -   about 10-50% [sugar acid anion][cation] (w/w).

In some embodiments, the ionic liquid solution contains:

-   -   about 50-90% (w/w) water; and    -   about 10-50% 1:2 [sugar acid anion][cation] (w/w).

In some embodiments, the ionic liquid solution contains:

-   -   about 50-90% (w/w) water; and    -   about 10-50% 1:2 [mucate di-anion][cation] (w/w).

In some embodiments, the ionic liquid solution contains:

-   -   about 50-90% (w/w) water; and    -   about 10-50% 1:2 [α-ketoglutarate di-anion][cation] (w/w).

In some embodiments, the ionic liquid solution contains:

-   -   about 50-90% (w/w) water; and    -   about 10-50% dicholine mucate (w/w).

In some embodiments, the ionic liquid solution contains:

-   -   about 50-90% (w/w) water; and    -   about 10-50% dicholine α-ketoglutarate (w/w).

The pretreatment mixture can contain any suitable amount ofpolysaccharide biomass. In general, the pretreatment mixture contains upto about 50% biomass by weight of the pretreatment mixture. Thepretreatment mixture can contain, for example, from about 0.1 to about50% biomass, or from about 5% to about 45% biomass, or from about 10% toabout 40% biomass, or from about 15% to about 35% biomass, or from about20% to about 30% biomass, or from about 5% to about 40% biomass, or fromabout 5% to about 30% biomass, or from about 5% to about 20% biomass, orfrom about 5% to about 10% biomass by weight of the pretreatmentmixture. The pretreatment mixture can contain about 1, 5, 10, 15, 20,25, 30, 35, 40, 45, or 50% biomass by weight of the pretreatmentmixture. In some embodiments, the mixture includes from about 5% (w/w)to about 30% (w/w) polysaccharide biomass. Other amounts of biomass canbe used in the methods of the invention, depending in part on factorssuch as the type of biomass material and the particular ionic liquidused in the method.

Biomass Pretreatment

Pretreatment of the polysaccharide biomass in the ionic liquid solutioncan be conducted for any suitable length of time at any suitabletemperature and pressure. In general, pretreatment is conducted foranywhere from a few minutes to several hours. Pretreatment can beconducted, for example, for about five minutes, or about 10 minutes, orabout 30 minutes, or about 60 minutes. Pretreatment can be conducted forabout 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 9, 12, 15, 18, 21, 24,36, 48, 60, or about 72 hours. Pretreatment is generally conducted at atemperature ranging from about 20° C. to about 200° C. Pretreatment canbe conducted, for example, at a temperature ranging from about 20 ° C.to about 100° C., or from about 40° C. to about 80° C., or from about100° C. to about 200 ° C., or from about 120° C. to about 180° C., orfrom about 140° C. to about 160° C., or from about 40° C. to about 180°C., or from about 60° C. to about 160 ° C., or from about 80° C. toabout 140° C., or from about 100 to about 120° C. Pretreatment can beconducted at about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, or about 200° C. for at least about0.5, 1, 3, 6, 9, 12, or 16 hours. Pretreatment can be conducted atatmospheric pressure or elevated pressures. Pretreatment can beconducted, for example, at a pressure (Pg) ranging from about 14 psi toabout 4000 psi, or from about 14 psi to about 3500 psi, or from about 14psi to about 2500 psi, or from about 14 psi to about 1500 psi. Incertain embodiments, the pretreatment is conducted at around atmosphericpressure (i.e., 14.696 psi).

In some embodiments, the invention provides a method for preparing asugar composition as described above wherein step ii) includesmaintaining the mixture of step i) at a temperature of at least about100° C. for at least about 30 minutes.

Biomass Saccharification

Following pretreatment of the polysaccharide biomass, the pH of themixture containing the dissolved polysaccharide and the ionic liquidsolution is reduced to a level that is suitable for enzymatic hydrolysisof the polysaccharide by one or more glycoside hydrolases. In general,the pH of mixture is reduced to at most about 7. The pH of the mixturecan be reduced, for example, to less than 7, less than 6.5, less than 6,less than 5.5, or less than 5. In certain embodiments, the pH of themixture is reduced to a pH of from about 5 to about 6.

The pH of the mixture containing the dissolved polysaccharide can bereduced by adding an acid to the mixture. Any suitable acid can be usedto reduce the pH. Suitable acids include, but are not limited to,hydrochloric acid, sulfuric acid, nitric acid, acetic acid, citric acid,formic acid, and the like. In certain embodiments, the acid used forreducing the pH of the mixture containing the dissolved polysaccharideis the same sugar acid that is present in the ionic liquid. In certainsuch embodiments, the mixture containing the dissolved polysaccharideand the ionic liquid solution is combined with one molar equivalent ofthe sugar acid (with respect to the amount of the sugar acid present inthe ionic liquid). As a non-limiting example, combination of oneequivalent of mucic acid with a mixture containing dissolvedpolysaccharide and dicholine mucate will reduce the pH of the mixture tobetween about 5 and about 6. Further adjustments to the pH can be madeby adding further amounts of acid (e.g., hydrochloric acid) to themixture as necessary. One of skill in the art will appreciate that thepH of the mixture containing the dissolved polysaccharide can beadjusted to maximize the activity of an enzyme, or a mixture of enzymes,e.g., one or more glycoside hydrolases, used in the subsequenthydrolysis step. The particular pH will depend in part on factorsincluding, but not limited to, the specific glycoside hydrolase(s) andthe amount of ionic liquid in the mixture.

Accordingly, some embodiments of the invention provide a method forpreparing a sugar composition as described above, which furthercomprises step ii-b): reducing the pH of the mixture containing thedissolved polysaccharide to less than 7 prior to adding the glycosidehydrolase in step iii). In some embodiments, step ii-b) includes addingan acid to the mixture resulting from step ii). In some suchembodiments, the acid used in step ii-b) is the same sugar acid used instep i). In some such embodiments, the molar amount of acid in stepii-b) is equal to the molar amount of acid in step i).

The methods of the invention generally include adding on or more enzymesthat break down polysaccharide biomass into smaller components.Typically, the pretreated biomass is subjected to the action of one, ormultiple, enzyme activities selected from a protease, a lipase, acellulase, an amylase, a glucano-hydrolase, a pectinase, a xylanase, aferulic acid esterase, and a mannanase. The pretreated biomass may alsobe treated with other enzymes, e.g., hemicellulases, that are used forthe degradation of biomass.

In some embodiments, the glycoside hydrolase is selected from anendoglucanase, an exoglucanase, a β-glucosidase, a xylanase, andmixtures thereof. In some embodiments, one or more cellulases are addedto the pretreated biomass present in the ionic liquid mixture in whichthe pH has been reduced, e.g., to at least about 7, following treatmentat a high pH.

A “cellulase” as used herein is a glycoside hydrolase enzyme thathydrolyzes cellulose (β-1,4-glucan or β-D-glucosidic linkages) resultingin the formation of glucose, cellobiose, cellooligosaccharides, and thelike. In the context of the present invention, cellulases includeendoglucanases; exoglucanases or cellobiohydrolases; and β-glucosidases.Endoglucanases (EC 3.2.1.4) including endo-1,4-β-glucanases or1,4-β-D-glucan-4-glucanohydrolases, act randomly on soluble andinsoluble 1,4-β-glucan substrates. Exoglucanases(exo-1,4-β-D-glucanases, e.g., the 1,4-β-glucan glucohydrolases; EC3.2.1.74) liberate D-glucose from 1,4-β-D-glucans and hydrolyzeD-cellobiose slowly. Cellobiohydrolases (1,4-β-D-glucancellobiohydrolases, EC 3.2.1.91) liberate D-cellobiose from1,4-β-glucans. β-Glucosidases ([β]-D-glucoside glucohydrolase;β-D-glucosidases; EC 3.2.1.21) act to release D-glucose units fromcellobiose and soluble cellodextrins, as well as an array of glycosides.Endoglucanases act mainly on the amorphous parts of the cellulose fiber,whereas cellobiohydrolases are also able to degrade crystallinecellulose.

A combination of two or more cellulases can be used in the methods ofthe invention. Cellulases act in concert to catalyze the hydrolysis ofcellulose-containing substrates. For example, endoglucanases breakinternal bonds and disrupt the crystalline structure of cellulose,exposing individual cellulose polysaccharide chains (“glucans”).Cellobiohydrolases incrementally shorten the glucan molecules, releasingmainly cellobiose units (a water-soluble β-1,4-linked dimer of glucose)as well as glucose, cellotriose, and cellotetrose. β-glucosidases splitthe cellobiose into glucose monomers. The cellulase can be athermostable cellulase. In certain embodiments the glycoside hydrolase,such as a cellulase, is selected such that it can perform optimally inthe presence of ionic liquid.

A xylanase and/or a “mannanase” may also be employed in thesaccharification of pretreated biomass. A “xylanase” is a glycosidehydrolase enzyme that catalyzes the endo-hydrolysis of 1,4-β-D-xylosidiclinkages in xylans. Xylanases include enzymes classified as a1,4-β-D-xylan-xylohydrolase (E.C. 3.2.1.8).

A “mannanase” is a glycoside hydrolase that hydrolyzes1,4-β-D-mannosidic linkages in mannans, galactomannans and/orglucomannans. “Mannanase activity” refers to hydrolysis of1,4-β-D-mannosidic linkages in mannans, galactomannans and/orglucomannans. Mannases include enzymes classified as EC 3.2.1.78.

Cellulases suitable for use in the present invention are commerciallyavailable from, for example, Genencor (USA) and Novozymes (Europe). Forinstance, Novozyme has a number of different enzymes and enzymecomplexes that are specifically designed to be useful for the hydrolysisof lignocellulosic materials. Examples include, but are not limited to,the following: NS50013, which is a cellulase; NS50010, which is aβ-glucosidase; NS22086, which is a cellulase complex; NS22086, which isa xylanase; NS22118, which is β-glucosidase; NS22119, which is an enzymecomplex of carbohydrases, including arabinase, β-glucanase, cellulase,hemicellulase, pectinase, and xylanase; NS22002, which is a mixture ofβ-glucanase and xylanase; and NS22035, which is a glucoamylase. Inaddition, suitable thermostable cellulases are disclosed inInternational Pat. Appl. Pub. No. WO 2010/124266, which is incorporatedherein by reference. Other hydrolases suitable for hydrolyzing thepretreated biomass, i.e., the lignocellulosic material, will be known tothose of skill in the art. See e.g., Viikari et al., Adv. Biochem. Eng.Biotechnol., 108:121-45, 2007; and U.S. Pat. Appl. Pub Nos. US2009/0061484; US 2008/0057541; and US 2009/0209009, which areincorporated herein by reference.

Any suitable amount of enzyme or enzyme mixture, e.g., glycosidehydrolase or mixture of glycoside hydrolases, can be used in the methodsof the invention. In general a sub-stoichiometric amount of theglycoside hydrolase, with respect to the dissolved polysaccharide, isused. The amount of glycoside hydrolase can be expressed as activityunits. Alternatively, the amount of the glycoside hydrolase used in themethods of the invention can be expressed relative to the amount ofbiomass treated in the pretreatment step.

For example, the hydrolysis mixture can contain a glycoside hydrolase(or a mixture of glycoside hydrolases) in an amount ranging from about0.01 to about 10% (w/w), with respect to the amount of biomass used inthe pretreatment step. Thus, for example, when the method is conductedusing 1 kg of biomass, for example, the hydrolysis step can be conductedwith a glycoside hydrolase or a mixture of glycoside hydrolases in anamount ranging from about 100 mg to about 100 g. Those of skill in theart will appreciate that the amount of glycoside hydrolase or mixture ofenzymes used in the methods of the invention will depend in part onfactors including, but not limited to, the particular enzyme used, thenature of the biomass source, and the extent of the pretreatment step.

The enzymatic hydrolysis step can be conducted for any length of time atany suitable temperature. The enzymatic hydrolysis step can beconducted, for example, for about 2, 5, 10, 15, 30, 45, or 60 minutes.The enzymatic hydrolysis step can be conducted for about 1, 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 5.5, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, or 72hours. Enzymatic hydrolysis is generally conducted at a temperatureranging from about 20° C. to about 60° C. Enzymatic hydrolysis can beconducted, for example, at a temperature ranging from about 20° C. toabout 40° C., or from about 40° C. to about 60° C. Enzymatic hydrolysiscan be conducted at about 25° C., about 37° C., or about 55° C. for atleast about 10, 20, 30, 60, or 90 minutes or for at least about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 24, 48, or 72 hours.

Sugar Compositions

The methods of the invention provide sugar compositions containing oneor monosaccharides and/or oligosaccharides. Monosaccharides present inthe sugar compositions can include, but are not limited to, fucose,arabinose, rhamnose, galactose, mannose, xylose, glucose, glucuronicacid, and galacturonic acid. The oligosaccharides in the sugarcompositions contain monosaccharide subunits (e.g., fucose, arabinose,rhamnose, galactose, mannose, xylose, glucose, glucuronic acid, andgalacturonic acid) linked together via glycosidic bonds. Typically,between about 10% and about 100% conversion of the polysaccharidebiomass to sugars results from the methods of the invention. Thus, e.g.,processing of 1 kg of polysaccharide biomass according to the methods ofthe invention can yield from about from about 110 g to about 1100 g ofthe constituent monosaccharides and oligosaccharides in the final sugarcompositions. For example, processing of 1 kg of switchgrass accordingto the methods of the invention can yield sugar compositions containingfrom about 0.1 g to about 350 g of glucose and from about 0.1 g to about210 g to xylose. One of skill in the art will appreciate that thecomponents and the yield of the sugar composition will depend, in part,on the specific source of the biomass and the specific conditions thatare used for pretreatment and hydrolysis.

Sugar compositions obtained using the methods of the invention can beused as carbon sources for host cells to produce useful organiccompounds such as biofuels. The sugar compositions can be used for theproduction of fermentation products (e.g., ethanol, tartaric acid,itaconic acid, succinic acid, propanediol, butanol, glycerol, and thelike) as described, for example, in International Pat. Appl. Pub. No. WO2016/070125, which is incorporated herein by reference.

Ionic Liquid Recycling

The methods of the invention allow for simple recycling of the ionicliquid from one form to another for re-use. This is particularly truefor embodiments wherein the same acid (e.g., mucic acid) is used in thepretreatment step and the pH reduction step. The pretreatment step isperformed in the basic stoichiometry, and then the solution is acidifiedfor the enzymatic hydrolysis step. After the hydrolysis, the solublesugars can be removed by any of the previously reported methods. Forexample, if a liquid-liquid extraction with boronic acids is used, thesolution can be ‘switched’ back to the basic form first, which matchesthe conditions required for efficient extraction [Brennan, et al. 2010.BioEnergy Research 3: 123-33; Shi, et al. 2013. Green Chemistry 15:2579-89]. After extraction, the IL-phase can be recycled to pretreatanother batch of biomass. Alternatively, the hydrolysate can be feddirectly into the fermentation step. In certain embodiments, E. coli, ora yeast, e.g., S. cerevisiae, or other microorganisms can tolerate highconcentrations of the hydrolysate produced by this process, limiting thedilution required.

The biomass input, glucose and xylose output, and the insolublelignin/ash output can be measured for the methods of the invention, anda biomass mass balance for the pretreatment and saccharification stepscan be constructed from these streams. Nearly 76% of the originalbiomass leaves the reactor in the liquid stream.

III. EXAMPLES Example 1 Preparation of Choline Mucate Ionic Liquids anduse Thereof

In a typical process, choline hydroxide ([Ch][OH], 46 wt % in H₂O) istaken in a glass beaker with a magnetic stirrer, with continuousstirring. Mucic acid (40 wt % in H₂O) is added dropwise at roomtemperature until the pH of the resulting IL reaches 13.5. At thispoint, the resulting dicholine mucate ionic liquid (i.e., [Ch]₂[Mu])contains two molar equivalents of choline cations and one molarequivalent of mucate anions. [Ch]₂[Mu] (40 wt % in H₂O) is used forfurther pretreatment.

[Ch]₂[Mu] is then used for one-pot pretreatment and saccharification. Inan integrated process, 1 g switchgrass is mixed with 9 g [Ch]₂[Mu] (40wt % in H₂O) at a 10 wt % biomass loading in a 35 mL pressure tube andpretreated at 140° C. for 1 h. After pretreatment, the slurry is dilutedwith DI-water to obtain a final [Ch]₂[Mu] concentration of 20 wt %.Before adding enzyme mixture (CTec2/HTec2=9:1, v/v) forsaccharification, mucic acid (40 wt % in H₂O) is added to the slurry toreduce the pH of the system to 5. In some instances, citrate buffer canbe added into the system (e.g., at a concentration of 50 mM) to maintainthe mixture at the desired pH. Enzymatic hydrolysis is conducted at 50°C. in a 50 mL plastic tubes for 3 days with an enzyme loading of 10 mgprotein/g switchgrass. The glucose yield is 76.3% and xylose yield is49.6%.

FIG. 1 shows a comparison of sugar yield resulting from one-potpre-treatment and saccharification of switch grass using an aldaricacid-based choline ionic liquid, [Ch]₂[Mu], and using [Ch]₂[glutamate].Advantageously, the sugar compositions can be prepared in good yieldusing sugar-derived ionic liquids for biomass pretreatment andsaccharification. Because the ionic liquids can be prepared withinexpensive materials that are readily available at biorefineries, theexpense associated with non-lignocellulosic materials (e.g., glutamicacid-based ionic liquids) can be reduced or eliminated.

Example 2 Preparation of [Ch] [α-Kg] and use Thereof

In a typical process, choline hydroxide ([Ch][OH], 46 wt % in H₂O) ismixed with α-ketoglutaric acid (40 wt % in H₂O) at room temperatureuntil the pH reaches 13.5. At this point, the resulting dicholineketoglutarate ionic liquid (i.e., [Ch]₂[α-Kg]) contains two molarequivalents of choline cations and one molar equivalent of mucateanions.

[Ch]₂[α-Kg] is then used for one-pot pretreatment and saccharification.In an integrated process, 40 g corn stover (2 mm particle size) is mixedwith 160 g [Ch]₂[α-Kg] (40 wt % in H₂O) at a 20 wt % biomass loading ina 300 mL Parr reactor and pretreated at 120° C. for 4 h. Afterpretreatment, the slurry is diluted with DI-water to obtain a final ILconcentration of 10 wt %. Before adding enzyme mixture (CTec2/HTec2=9:1,v/v) for the saccharification, α-Ketoglutaric acid (40 wt % in H₂O) isused to drop and maintain the pH of the system to 5. Enzymatichydrolysis is conducted at 50° C. in a 1L shake flask for 3 days with anenzyme loading is 20 mg protein/g corn stover.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. An ionic liquid comprising at least one anion andat least one cation, wherein the anion is selected from the groupconsisting of a sugar acid anion and a ketoacid ion.
 2. The ionic liquidof claim 1, wherein the sugar acid is selected from the group consistingof an aldaric acid, an aldonic acid, a uronic acid, or a combinationthereof.
 3. The ionic liquid of claim 1, wherein the sugar acid isselected from the group consisting of mucic acid, saccharic acid,xylaric acid, arabinaric acid, and mannaric acid.
 4. The ionic liquid ofclaim 1, wherein the ketoacid is selected from the group consisting ofα-ketoglutaric acid, pyruvic acid, and levulinic acid.
 5. The ionicliquid of claim 1, wherein the anion is selected from the groupconsisting of a mucic acid anion, a mucic acid anion, an α-ketoglutaricacid anion, and an α-ketoglutaric acid dianion.
 6. The ionic liquid ofclaim 1, wherein the cation is selected from choline, (C₁₋₁₈ alkyl)₃NH⁺,(C₁₋₆ alkyl)_(x)(C₆₋₁₈ alkyl)_(y)N⁺, (C₁₋₁₀ alkyl)_(z)imidazolium,(C₁₋₁₈alkyl)_(z)pyrazolium, and mixtures thereof; wherein subscript xand subscript y are each 0, 1, 2, 3, or 4, and the sum of x and y is 4;and wherein each subscript z is 1, 2, or
 3. 7. The ionic liquid of claim6, wherein the cation is choline.